In Vitro Transporter Assays

It's crucial to understand your compound's potential for drug-drug interactions (DDI) by evaluating the compound's substrate and inhibition potential toward P-glycoprotein (P-gp) before investing in further preclinical development.

The FDA considers the bidirectional transport assay provided by Apredica as definitive for identifying P-gp substrates and inhibitors, as this assay measures drug efflux in a more direct manner than other possible methods. In this assay, a compound is considered to be a P-gp substrate if the calculated efflux ratio

Apredica recommends assessing P-gp transport in Caco-2 cell cultures because Caco-2 also permits other transporters to be investigated. As Apredica continuously cultivates Caco-2 cell cultures, the 21-day incubation period is not an impediment to turnaround. MDCK cell lines are also available. Transporter assays are available in the following variants:

• Bi-directional (P-gp substrate determination): Test agent is incubated on either side of the monolayer in the presence and absence of a known P-gp inhibitor.
• Bi-directional (P-gp inhibitor determination): Test agent is incubated on either side of the monolayer in the presence and absence of a known P-gp substrate.
• Bi-directional (Other transporters): Please inquire regarding the testing of your test agents to be substrates or inhibitors of other efflux transporters (e.g. BCRP, MRP1, MRP2, etc.). You may also request the poster we presented at the 16th North American Regional ISSX Meeting where we fully characterized activity of transporters in CaCo-2 cells.

ATP-binding cassette (ABC) transporters are involved in the disposition of endogenous compounds and xenobiotics. These transporters use ATP for energy. They can be inhibited or induced, which changes the bioavailability and disposition of drugs. The ABC transporter family includes:

• multidrug resistance proteins (MDR)
• multidrug resistance associated proteins (MRP)
• breast cancer resistance protein (BCRP)
• bile salt export pump (BSEP) ¹

Substantial clinical evidence has accrued to support a key role for the MDR1 gene product, also known as ABCB1, or more commonly, P-glycoprotein (P-gp), in incomplete, variable, and nonlinear absorption for a wide variety of drugs, such as cyclosporine, talinolol, tacrolimus, digoxin, fexofenadine, and saquinavir.² Clinical evidence suggests the involvement of P-gp in biliary and renal excretion and brain penetration of some drugs.^{3, 4}

P-gp is located in the apical/luminal membrane of polarized cells (i.e., enterocyte brush border membranes, hepatocyte canalicular membranes, renal proximal tubular cell luminal membranes, and endothelial cell membranes at the blood-brain barrier).^{5, 6} P-gp's broad substrate specificity and high transporter capacity produces the possibility of drug-drug interactions. A drug interacting with P-gp may inhibit the transport of a co-administered drug. This inhibition may be either competitive or noncompetitive, and may result in increased bioavailability, decreased clearance, or significantly elevated AUC of the affected drug. For example, erythromycin (a P-gp inhibitor) co-administered with talinolol (a P-gp substrate) produces a 34% increase in talinolol's AUC.⁷ Drug interactions due to P-gp induction are also possible. For example, cyclosporine (a P-gp inducer) significantly increases the clearance of fexofenadine (a P-gp substrate).⁸

References

1. Sahi J (2005) Use of In Vitro Transporter assays to understand hepatic and renal disposition of new drug candidates. Exp Op Drug Metab1:409-427.

2. Hoffmann U, Kroemer HK (2004) The ABC transporters MDR1 and MRP2: multiple functions in disposition of xenobiotics and drug resistance. Drug Metab Rev 36:669-701.

3. Kullak-Ublick GA, Becker MB (2003) Regulation of drug and bile salt transporters in liver and intestine. Drug Metab Rev 35:305-317.

4. Ieiri I, Takane H, Otsubo K (2004) The MDR1 (ABCB1) gene polymorphism and its clinical implications. Clin Pharmacokinet 43:553-576.

5. Mealey KL (2004) Therapeutic implications of the MDR-1 gene. J Vet Pharmacol Ther 27:257-264.

6. Tanigawara Y (2000) Role of P-glycoprotein in drug disposition. Ther Drug Monit 22:137-140.

7. Schwarz U, Grammate T, Krappweis J et al. (2000) P-glycoprotein inhibitor erythromycin increases oral bioavailability of talinolol in humans. Int J Clin Pharmacol Ther 38:161-167.

8. Dresser G, Schwarz U, Wilkinson G, Kim R (2003) Coordinate induction of both cytochrome P4503A and MDR1 by St John's wort in healthy subjects. Clin Pharmacol Ther 73:41-50.